1. Field of the Invention
The present invention relates generally to a method of inducing, stimulating or otherwise facilitating bronchoprotection in humans and animals by modulating bronchial constriction and/or inflammation. The present invention is predicated in part on the identification of receptors in airway epithelium which mediate inhibition of bronchoconstriction and/or inflammation following their activation. More particularly, the present invention identifies that activation of protease activated receptors (PARs) results in relaxation of airway epithelium. Activation of airway epithelium PARs inhibits bronchoconstriction and/or inflammation and thereby mediates bronchoprotection of the airways. The present invention further provides a method for the prophylaxis and treatment of disease conditions in airways such as asthma and bronchitis and further provides methods for the diagnosis and screening of agents useful in the prophylaxis and treatment of airway disease conditions.
2. Description of the Related Art
Many receptors for biologically-active effector molecules are large proteins embedded in biological membranes. They serve as transducers of information mediated by effectors such as hormones and cytokines, and are also important in the mechanism of action of pharmaceutical agents. For example, receptors located within the outer regions of the cellular membrane act to transduce such information into the cell, which may then respond in a number of different ways via specific secondary messenger systems. Therefore, these types of receptors have specific extracellular and intracellular domains which allow information, such as hormonal signals, to be appropriately detected and processed by cells.
Protease-activated receptors (PARs) are a relatively new subtype of a superfamily of membrane receptors which have seven membrane-spanning regions and are coupled to intracellular second messenger mechanisms via G proteins. The three known members, respectively designated PAR1, PAR2 and PAR3, have been cloned, and shown to be expressed in vascularised tissues comprising endothelial and smooth muscle cells (PAR1 and PAR2) and platelets (PAR1 and PAR3). A fourth receptor, designated PAR4, has also recently been demonstrated on platelets of PAR3 deficient mice and has been cloned (Kahn et al, 1998); the human homologue has also been cloned (Xu et al, 1998).
PARs are activated in a unique manner, which is illustrated in FIG. 1. As the name indicates, limited proteolysis by specific proteases (proteinases) removes part of the extracellular N-terminal region of the receptor, so that the newly-shortened N-terminal acts as a ligand for an as yet undefined binding region on the remainder of the receptor in order to signal the cell to respond. Thus, PARs have their own inbuilt or “tethered” ligands, and the specific protease activity reveals that these latent, intrinsic ligands act as ligands in their own right rather than as exogenous effectors.
PAR1 (Vu et al, 1991; Coughlin et al, 1992) and PAR3 (Ishihara et al, 1996) are activated primarily by the blood-borne protease, thrombin, which is believed to be involved in thrombosis, inflammation and mitogenic growth (De Catering & Sicari, 1993; Dennington & Berndt 1994; Fager, 1995). For example, thrombin causes smooth muscle in the airways to proliferate, which may cause the airway to thicken and become obstructed. PAR1 is also located on vascular endothelial cells, where, like many other receptor types, stimulation leads to release of nitric oxide (NO) and other factors which then cause the muscle in the wall of the vessels to relax (Muramatsu et al, 1991; Tesfamariam et al, 1993; Tesfamariam, 1994; Hwa et al, 1996; Saifeddine et al, 1996). Under normal circumstances, the enzymatic activity of thrombin is strongly suppressed by a number of endogenous inhibitors.
PAR2 differs from both PAR1 and PAR3 receptors in that it is activated not by thrombin, but by trypsin and trypsin-like enzymes, such as mast cell-derived tryptase (Molino et al, 1997). Trypsin is usually confined to the upper gastrointestinal tract after its generation by activation of its pancreatic precursor, trypsinogen. Trypsinogen is induced in vascular endothelial cells by tissue plasminogen activator [TPA] (Koshikawa et al, 1997). Tryptase is released in large concentrations from mast cells (Caughey, 1994). Mast cells are believed to have a central role in the pathogenic manifestations of asthma. Tryptase stimulates mucus release and can inactivate some peptides such as vasoactive intestinal peptide (VIP) that relax airway smooth muscle in experimental animals. This suggests that the PARs play a role in the aetiology of airway disease by inducing contraction of smooth muscle cells.
In addition to tryptase, tryptase-like enzymes are released by clara cells (Yasuoka et al, 1997), which are common in the epithelium lining the small bronchi of most mammals, including humans, the trachea of the mouse, and by lymphocytes which enter the inflamed airway in large numbers. Trypsin has been localised to normal airway epithelium (Koshikawa et al, 1997). In addition, tryptase-like enzymes are thought to be involved in a number of inflammatory responses and diseases, such as atherosclerosis (Atkinson et al, 1994; Kovanen et al, 1995) and varicosis (Yamada et al, 1996). Furthermore and importantly, as well as directly activating mast cell degranulation via IgE-antigen recognition, the antigens of some dust mites and pollens are proteases with trypsin-like activity (Caughey, 1997). Therefore, allergens which are central to, and the causal agents of, many airway diseases have the potential to directly and indirectly activate PAR2.
PAR1 and PAR2, but not PAR3 (Isihara et al, 1997) can also be activated by short synthetic peptide sequences corresponding to those of the tethered ligands. For PAR1, this tethered ligand is SFLLRN—NH2 (SEQ ID NO:1), which is also known as TRAP (thrombin receptor-activating peptide)). The tethered ligand sequence for mouse PAR2 is SLIGRL—NH2 (SEQ ID NO:2), and is referred to herein as PAR2 activating peptide (PAR2-AP). Therefore, these peptides can be used to mimic enzyme mediated PAR activation and to study the effects of PAR activation.
The genes for PAR1, PAR2 and PAR3 have been cloned (Vu et al, 1991; Nystedt et al, 1994; Bohm et al, 1996a; Saifeddine et al, 1996 and Ishihara et al, 1997). PAR2 mRNA has been shown to be highly expressed in vascularised or endothelialised tissues such as the stomach, intestine, pancreas, kidney and liver. In the gut, PAR2 mRNA is located mainly in epithelial cells (Bohm et al, 1996b). In blood vessels, functional PAR2 has been localised nearly exclusively to endothelial cells, where, like PAR1, it mediates endothelium-dependent vasodilation (Hwa et al, 1996; Saifeddine et al, 1996). It has been proposed that PAR2 acts as a trypsin sensor in the pancreas (Bohm et al, 1996a) and is involved in a possible cytoprotective mechanism for gut epithelia exposed to trypsin (Bohm et al, 1996b). Apart from these proposed activities, little is known of other physiological roles for these receptors.
Following activation, PARs are inactivated by rapid internalization, which also provides the signals for rapid generation of new receptors from intracellular pools and de novo protein synthesis (Hoxie et al, 1993; Bohm et al, 1996b). This provides a powerful self-replenishing system to maintain adequate tissue levels of receptors.
Like PAR1, PAR2 mediates relaxation of arteries via the release of nitric oxide (NO; Moncada et at, 1991) and of endothelium-derived hyperpolarising factor (EDHF: Garland et al, 1995), although the EDHF-dependent mechanism for PAR1 is different from that for PAR2. The mechanisms of receptor recycling also regulate the way in which endothelial cells recover their ability to respond to further protease challenge, at least within two to three hours after the first challenge. For PAR1, this recovery process involves rapid recycling of receptors (30 min–150 min) without the tethered ligand sequence, but no new N-terminal receptors are produced. For PAR2, however, fully intact new receptors are rapidly synthesized from stable mRNA, and are inserted into the plasma membrane (Bohm et al, 1996a).
Only PAR1 has been identified in the human vasculature (Nelken, 1992), where expression was reported to be isolated to endothelial cells in atheroma-free arteries. In vessels affected by atherosclerosis, PAR1 mRNA was found in endothelial, smooth muscle and mesenchymal-appearing cells. Studies on human endothelial cell PAR function have been limited to the measurement of calcium fluxes in transfected cell lines (Marl, 1995) and umbilical vein endothelial cells (Ngaiza et al, 1991; Kruse et al, 1995). An atypical PAR has also been identified in human coronary arteries (Hamilton et al, 1998).
The incidence and prevalence of airway diseases such as asthma and bronchitis, which are characterized by airflow obstruction, inflammation and pathological changes in airway tissue are increasing globally (Barnes et al, 1996a). However, it is unknown why some people develop these types of airway diseases, while other people exposed to the same environmental factors do not. One possibility is that the airway defenses of patients who develop the disease are less efficient than those of non-afflicted subjects.
Asthmatic patients suffer from episodic airflow limitation caused by bronchospasm, oedema and thickening of the airway walls. In addition, one of the hallmarks of asthma is that the bronchi are hypersensitive to specific and non-specific stimuli, causing them to contract too much and too sensitively, thereby narrowing the airways and making breathing difficult (Barnes, 1996b; Barnes et al, 1996c). The most widely-used treatment for asthma is administration of drugs that cause the bronchial muscles to relax and the airways to dilate, thus restoring the ability to breath. The most commonly used drugs for this purpose are the so-called beta-2 agonists. These drugs stimulate another subtype of the seven transmembrane, G protein-coupled receptor superfamily, the beta-2 adrenoceptors, which are located on the muscle and mediate relaxation via well-defined biochemical mechanisms. While beta-2 agonists are effective in most patients, it has recently been discovered that some asthmatics respond poorly to beta-2 agonists, and the agonists may mediate down-regulation of patient responses during chronic treatment due to genetic mutations in the beta-2 adrenoceptor sequence. Additionally, concerns have been raised about the possibility that regular use of beta 2-adrenoceptor agonists may increase the risk of death from asthma.